The presence of Alpha2-adrenergic receptors was investigated in smooth muscle cell cultures using 3H clonidine (Alpha2-adrenergic agonist) as a ligand. Specific binding sites of 3H clonidine were defined as the excess over "blank" taken in the presence of 1Mum "cold" clonidine. For the competitive studies different concentrations of various adrenergic agonists and antagonists were used to displace binding with 4nM 3H-clonidine. The rank order of potency for Alpha-adrenergic agonists and antagonists was clonidine greater than phentolamine = yohimbine greater than greater than prazosin. The IC50 for the investigated displacers were: 25nM, 300nM, 1Mum and 9mM, respectively. Competition curve produced by competing "cold" for 3H-clonidine (4nM) showed a biphasic pattern indicative of multiple binding sites. Besides, the Scotchard analysis of saturation curve (concentration of 3H-clonidine ranged from 6MuM to .3 nM) and dissociation rate were characteristic of multiple population of the alpha 2-adrenergic binding sites in cultured smooth muscle cells. Thus, the existence of Alpha2-type adrenergic receptors not linked to AC activity observed in the cerebrovascular smooth muscle cells strongly suggest that their reactivity which is mediated by these receptors might be associated with Ca++ fluxes.